Lactic acid bacteria are used extensively as starter cultures in the food industry in the manufacture of fermented products including milk products such as e.g. yoghurt and cheese, meat products, bakery products, wine and vegetable products. Lactococcus species including Lactococcus lactis are among the most commonly used lactic acid bacteria in dairy starter cultures. However, several other lactic acid bacteria such as Leuconostoc species, Pediococcus species, Lactobacillus species and Streptococcus species. Species of Bifidobacterium, a group of strict anaerobic bacteria, are also commonly used in food starter cultures alone or in combination with lactic acid bacterial species.
When a lactic acid bacterial starter culture is added to milk or any other food product starting material under appropriate conditions, the bacteria grow rapidly with concomitant conversion of citrate, lactose or other sugar compounds into lactic acid/lactate and possibly other acids including acetate, resulting in a pH decrease. In addition, several other metabolites are produced during the growth of lactic acid bacteria. These metabolites include ethanol, formate, acetaldehyde, α-acetolactate, acetoin, diacetyl, and 2,3 butylene glycol (butanediol). Among these metabolites, diacetyl is an essential flavour compound which is formed during fermentation of the citrate-utilizing species of e.g. Lactococcus, Leuconostoc and Lactobacillus. Diacetyl is formed by an oxidative decarboxylation (FIG. 1) of α-acetolactate which is formed by the action of α-acetolactate synthetase (Als) from two molecules of pyruvate. Pyruvate is a key intermediate of several lactic acid bacterial metabolic pathways including the citrate metabolism and the degradation of lactose or glucose to lactate. The pool of pyruvate in the cells is critical for the flux through the metabolic pathway leading to diacetyl, acetoin and 2,3 butylene glycol (butanediol) via the intermediate compound α-acetolactate due to the low affinity of α-acetolactate synthetase for pyruvate.
Pyruvate is converted to formate and acetyl coenzyme A (acetyl CoA) (FIG. 1) by the action of pyruvate formate-lyase (Pfl). This conversion takes place only under anaerobic conditions (Frey et al. 1994). Pfl is inactivated even at low levels of oxygen, and a switch from anaerobic to aerobic conditions will lead to significant changes in metabolic end product profiles in lactic acid bacteria with complete disappearance of ethanol and formate (Hugenholtz, 1993). Another factor which regulates the activity of Pfl is the pH. The pH optimum of Pfl is about 7 (Hugenholtz, 1993).
An alternative pathway for the formation of acetyl CoA from pyruvate (FIG. 1) in a lactic acid bacterium is by the activity of the pyruvate dehydrogenase complex (PDC). In contrast to Pfl, PDC has a very low activity under anaerobic conditions due to the inhibitory effect of NADH on that enzyme (Snoep et al. 1992). This enzyme requires the presence of lipoic acid as a co-factor to be active.
Additionally, acetyl CoA can be produced in lactic acid bacteria from acetate under aerobic as well as under anaerobic conditions.
Accordingly, it is conceivable that the pyruvate pool is increased under anaerobic conditions if the lactic acid bacterial strain is defective in enzyme systems involved in pyruvate consumption, including Pfl. As mentioned above, an increased pyruvate pool may lead to an increased flux from pyruvate towards acetoin and diacetyl or other metabolites derived from α-acetolactate. Thus, it is to be expected that fermented food products which are produced by using a lactic acid bacterial starter culture having a reduced Pfl activity or completely lacking such activity contain an increased amount of acetoin or other of the above metabolites. Conversely, the such starter cultures may produce reduced amounts of other metabolites, including ethanol and acetate and possibly, acetaldehyde.
Recent studies have shown that when L. lactis is lacking the lactate dehydrogenase (Ldh) which is involved in the major pyruvate consuming pathway leading to lactate, more pyruvate is directed towards acetoin and butanediol via α-acetolactate, possibly resulting in increased formation of the intermediate product diacetyl (Platteeuw et al., 1995; Gasson et al., 1996).
Overproduction of α-acetolactate synthetase in Lactococcus lactis as another approach of metabolically engineering lactic acid bacteria to produce increased amounts of diacetyl has been disclosed by Platteeuw et al. 1995.
The potential of using L. lactis strains with reduced pyruvate formate-lyase activity as a means of increasing diacetyl formation is mentioned by Hugenholtz, 1993. It is suggested by this author that the combination of three strategies: 1) Ldh inactivation by mutation/genetic engineering, 2) Pfl inactivation by aeration and/or low pH and 3) acetolactate decarboxylase (ALD) inactivation by mutation/genetic engineering could result in a high production of α-acetolactate from lactose.
However, the suggested inactivation of Pfl activity by aeration and/or low pH is not feasible or possible in the industrial production of lactic acid bacterially fermented dairy products or other fermented food products, as the production hereof generally takes place under essentially anaerobic conditions. Furthermore, the pH of the starting materials including milk is typically about 7 and it is generally not desirable to lower the pH of the food material to be fermented.
Whereas it has been suggested to modify the Pfl activity of lactic acid bacteria as a means of changing their production of metabolites in a desirable direction by manipulating the growth conditions, there have been no suggestions in the prior art to utilize metabolically engineered lactic acid bacteria which have a modified Pfl activity under industrially appropriate and feasible culturing conditions.
A method that allows isolation of mutants of gram-negative bacteria devoid of Pfl activity has been disclosed by Pascal et al., 1974. This method includes the selection of Pfl defective mutants of E. coli and Salmonella typhimurium based on their lack of ability to generate H2 and CO2 in the absence of formate, when they are incubated under anaerobic condition in media containing glucose or pyruvate. However, such a selection method cannot be used for selection of Pfl defective mutants of lactic acid bacteria, since these organisms lack the enzyme that catalyses production of H2 and CO2 from formate.
Accordingly, the prior art does not contain any guidance with respect to designing a feasible method of isolating a lactic acid bacterial Pfl defective (Pfl−) mutant.
Experiments performed by the inventor with the minimal medium BA (Clark and Maaløe, 1967) for E. coli, showed that this medium did not support the aerobic growth of lactic acid bacteria. However, if cultivated in this medium together with E. coli the growth of lactic acid bacteria was supported, indicating that E. coli produces a factor needed for the growth of the lactic acid bacteria. It has later been found that this growth factor is acetate, which led to the development of the DN-medium (Dickely et al., 1995).
It has now surprisingly been found that wild-type strains of lactic acid bacteria such as strains of Lactococcus and Streptococcus including as examples Lactococcus lactis and Streptococcus thermophilus strains under anaerobic conditions grow well on the DN-medium (Dickely et al., 1995) in the absence of acetate. These unexpected findings have made it possible to develop a novel and simple method for the isolation of Pfl defective lactic acid bacterial mutants based on the finding that such mutants, in contrast to the phenotypically Pfl+ wild-type strains, are unable to grow under anaerobic conditions on DN-medium in the absence of acetate.
Additionally, having such a method allowing the selection of Pfl defective lactic acid bacterial mutants at hand has made it possible to provide further mutated cells which in addition to being Pfl− are mutated in one or more genes involved in the citrate/sugar metabolic pathways such as e.g. the ldh gene coding for lactate dehydrogenase (Ldh) so as to provide a variety of metabolically engineered lactic acid bacteria having highly desirable improved characteristics with respect to metabolite (fermentation end product) production.
The above findings have thus opened up for a novel approach for providing useful metabolically engineered lactic acid bacterial starter cultures which approach is based on relatively simple classical random mutagenesis methods or the selection of spontaneously occurring mutants and which does not involve in vitro genetic engineering. From a practical technological point of view this is advantageous, since in most countries the use of genetically engineered food starter cultures is still conditional on approval by regulatory bodies.